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Human Journals

Research Article

October 2017 Vol.:7, Issue:4

© All rights are reserved by Sangavi. S et al.

Chromatography Profile from the Methanol Extract of

Abelmoschus esculentus L Flowers

www.ijsrm.humanjournals.com

Keywords: Abelmoschus esculentus, HPTLC, LCMS.

ABSTRACT

Herbal medicine is as primordial as mankind and has

appreciably contributed to the health care of the human

society. The study is to develop the high performance thin

layer chromatography (HPTLC) fingerprint profile of

Abelmoschus esculentus (L). HPTLC method for the

separation of the active constituents in extracts has been

developed using solvent system Toluene: Ethyl acetate:

Formic acid (5:4:1). In HPTLC analysis, it showed the

presence of quercetin in standard as well as the sample with

Rf value 0.56. The fingerprinting images will be helpful in

the identification and quality control of the drug and ensure

therapeutic efficacy. The methanol extracts of Abelmoschus

esculentus (L) flowers showed the presence of molecules

such as Protocatechuic Acid-4-o- Glucoside, Kaempferol-3-

o-Hexoside, Isorhamnetin-3-0- Glucoside, Quercetin Dimer

and Quercetin derivative by LCMS. Liquid chromatography-

mass spectrometry (LCMS) is proved to be a very useful

technique for plant metabolite and allows the identification of

a large variety of common plant metabolites in a single

chromatogram.

Sangavi. S*, Suja Pandian. R

PG and Research Department of Biochemistry,

Rabiammal Ahamed Maideen College for Women,

Thiruvarur, 610001, Tamilnadu, India

Submission: 25 September 2017

Accepted: 3 October 2017

Published: 30 October 2017


Citation: Sangavi. S et al. Ijsrm.Human, 2017; Vol. 7 (4): 93-106.

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INTRODUCTION

Almost 25 centuries ago, Hippocrates, the Father of Medicine, stated, “Let food be the

medicine and let medicine be the food”. Amidst ancient civilisations, India has been known

to be rich repository of medicinal plants [1]. Today a number of chemicals obtained from

plants are used as vital drugs in more countries in the world [2]. This knowledge is accessible

from thousands of medical texts and manuscripts. The substances having medical value have

been extensively used for treating various disease conditions. Herbs being easily available to

human beings have been explored to the maximum for their medicinal properties. Products of

primary metabolism such as amino acids, carbohydrates and proteins are vital for the

maintenance of life processes, while others like alkaloids, phenolics, steroids, terpenoids are

products of secondary metabolism and have toxicological, pharmacological and ecological

importance [3]. Many medicinal plants, traditionally used for thousands of years, are present

in a group of herbal preparations of the Indian traditional health care system (Ayurveda) and

proposed for their interesting multilevel activities. Amongst the medicinal plants used in

Ayurvedic preparations for their therapeutic action, some have been thoroughly investigated

and some need to be explored [4]. Ayurveda, Unani, Siddha and Folk (tribal) medicines are

the major systems of indigenous medicines. Among these systems, Ayurveda and Unani

Medicine are most developed and widely practiced in India [5].

Abelmoschus esculentus. (L) is an important medicinal plant of tropical and subtropical India.

Its medicinal usage has been reported in the traditional systems of medicine such as

Ayurveda, Siddha and Unani. Abelmoschus esculentus. (L) is a flowering plant in the mallow

family. Abelmoschus esculentus. L plays an important role in the human diet by supplying

carbohydrates, minerals, and vitamins and its flower have been consumed as health tea and

herbal medicine for hundreds of years. It is reported to have many curative effects, such as

antioxidant, anti-inflammatory and antitumor activities [6].

High performance liquid chromatography (HPLC) and Liquid chromatography mass

spectrography (LCMS) profiles were also recorded for which the plant extracts act as a

fingerprint for future investigations. HPTLC allows for the separation of the chemical

compounds that possess varying polarities thereby allowing for the profiling of the

compounds found in the plant species and thus serving as a fingerprint for the chemical

constituents of the plant species. HPTLC and LCMS analysis of plant species provide a



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means for the correct identification of the plant species and also to profile the chemical

composition of the plants [7].

MATERIALS AND METHODS

Flower sample collection

The fresh flower samples were collected in the month of October, from in and around

Nagapattinam, Nagapattinam district, Tamil Nadu, India. The flower samples were

thoroughly washed under running tap water to remove adhering dust particles and blotted dry

under shade for about two weeks, ground into milled powder and stored in an airtight

container used for further investigations.

Preparation of extracts

The powdered Abelmoschus esculentus (L) flower samples (1000g) were weighed and mixed

with 2000 ml of methanol. Then it is kept in an orbital shaker at 190-220 rpm for 48 hours.

The supernatant was collected, filtered through Whatman No.1 filter paper and then

concentrated by evaporating to dryness which gave a solid amorphous residue and it was

dried thoroughly to remove the solvent used. The obtained dried extract was then accurately

weighed, stored in small vials and used for the subsequent studies.

High performance thin layer chromatography (HPTLC) Analysis

Instrument : CAMAG Automatic TLC Sampler 4(ATS4) with win

CATS software.

Stationary phase : Plates silica gel 60 F 254 pre coated layer

Mobile phase : Toluene: Ethyl acetate: Formic acid (5:4:1)

Standard : Quercetin

Sample : Brown powder

Solubility : Methanol Standard concentration 1μl/ml

Standard preparation : Weigh 5mg of standard Quercetin in 5ml of methanol



96

Standard injection volume : 1, 2, 4, 6, 8, 10,12, 14, 16, 18μl

Sample concentration : 5, 10μl

Weigh about 100mg of

Hydro-alcoholic extract : Dissolved in 5ml of 7:3 Alcohol: Water

and Sample preparation

Sample application volumes : 1, 2,4,8,12,16

(μl)

Development mode : Ascending mode

Scanning wavelength : 254 nm and 366nm

Measurement mode : Absorbance

Evaluation: A band Rf value of 0.61 corresponding to Quercetin is visible in test solution

tracks

Preparation of the plates

The plates used for HPTLC was silica gel 60 F 254 (E.MERCK KGaA). 0.5µl to 3.5µl of

standard solution was applied in the form of bands using LINOMAT IV applicator. The

sample concentration was 10mg/ml and the different volumes were 10 to 50µl. The mobile

phase used was toluene: ethyl acetate: formic acid: methanol (5.5:3:1:0.5). The

chromatograph was developed for 15 minutes, dried at room temperature and scanned at

254nm and 366nm.

Procedure

Applied 1μl of standard solution and 5, 10μl of test solution on a percolated silica gel

60F254TLC Plate of uniform thickness 0.2mm using sample applicator. The plate was

developed in the solvent system to a distance of 8cm. The plate was scanned

densitometrically at 254 nm using TLC Scanner. The plate was observed under UV light at

254nm and 366nm using CAMAG REPROSTAR 3.



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Liquid chromatography is a fundamental separation technique used in life sciences and

related fields of chemistry. Liquid chromatography (LC) combined with mass spectrometry

(MS) is a powerful tool for qualitative and quantitative analytics of organic molecules from

various matrices, and the use of this hyphenated technique is very common in bioanalytical

laboratories. In the present study, LC/MS methods and the required sample preparation

applications were developed for detection of compounds such as flavones, terpene and

sesquiterpenes lactones.

Methanolic extract was used for the analysis. For determination of secondary metabolites,

micro TOF-Q II (Bruker, Germany), UV detector at 330nm and Quadrupole II for mass

analysis and TOF for mass detection was utilized. The column used was UHPLC Dionex C18

RP Acclaim 120Å, 2.1 × 150mm, 3.0μm column (Dionex, USA). Solution A: ACN (1%

Acetic acid) and Solution B: Water (1% Acetic acid) was used as mobile phase [8].

RESULTS

The study revealed that Abelmoschus esculentus. (L) flower showed best results in Toluene:

Ethyl Acetate: Formic acid 5:4:1 solvent system for methanolic extracts. After scanning and

visualizing the plates in absorbance mode at both 254nm, 366 nm and visible light range

(400-600nm after spraying with anisaldehyde sulphuric acid reagent) best results were shown

at 375nm. The HPTLC images shown in Figure 4 indicate that all sample constituents were

clearly separated without any tailing and diffuseness.

The results from HPTLC fingerprint scanned at wavelength 375nm for methanol extract of

Abelmoschus esculentus. L flower revealed the presence of seven polyvalent

phytoconstituents in standard Table 1 and five polyvalent phytoconstituents in sample (Table

2). The Rf values ranged from standard 0.62 to 0.60 and Rf values ranged from sample 0.64

to 0.63. It is also clear from Table 1 and Table 2 and the chromatogram as shown in Figure 2

and 3 that out of 12 components, the component with sample Rf values 0.63, 0.63 were found

to be more predominant as the percentage area is more with 100.00% and 100.00%

respectively. TLC plate showed different colour phytoconstituents of Abelmoschus

esculentus. (L) flower methanol extract (Track 1-2). The bands revealed presence of one blue,

light blue, three greenish, purple, one red and two light yellowish orange bands showing the

presence of steroids, terpenoids and saponins (Figure 4) after spraying with anisaldehyde

sulphuric acid reagent.



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The identity of the quercetin bands in sample chromatograms was confirmed by the

chromatogram obtained from the sample with that obtained from the reference standard

solution. HPTLC analysis of methanol extract of Abelmoschus esculentus. (L) flower was

carried out along with the standard quercetin and toluene: ethyl acetate: formic acid:

methanol (5.5:3:1:0.5) as the mobile phase. The identity of the bands of quercetin in the

methanol extract was confirmed by comparing the UV-Vis absorption spectra with those of

standards using a CAMAG TLC scanner3 (Figure 1). The standard quercetin has Rf value of

0.56 for quercetin (Table 1 and Figure 2). The 3-D spectrum of all tracks scanned at 254nm is

shown (Figure 4).

QUANTIFICATION OF QUERCETIN BY PHOTO DOCUMENTATION UNDER UV

HPTLC plate seen at 254 nm HPTLC plate seen at 366nm

Figure 1: Chromatogram after derivatization, A) Under UV 254nm B) Under UV

366nm.

PEAK DISPLAY (0.5µl of Standard) PEAK DISPLAY (1µl of Standard)




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Figure 2: Chromatogram of Standard Quercetin (0.5μl to 3μl of standard)

Table 1: Shows peak table with Rf values, height and area of Quercetin sample in

methanol extract of Abelmoschus esculentus. (L) flower

Peak Standa

rd (µl )

Start

Rf

Start

Height

Max

Rf

Max

Height

Height

%

End

Rf

End

Hei

ght

Area Area

%

1. 0.5 0.57 0.0 0.60 119 100.00 0.62 0.7 166.8 100.0

2. 1.0 0.55 1.2 0.59 418 100.00 0.61 0.1 668.0 100.0

3. 1.5 0.55 0.9 0.59 100.4 100.00 0.61 0.2 1510.7 100.0

4. 2.0 0.55 1.6 0.58 206.8 100.00 0.61 0.5 3041.3 100.0

5. 2.5 0.55 9.6 0.58 292.4 100.00 0.60 1.4 4289.2 100.0

6. 3.0 0.55 4.3 0.58 352.0 100.00 0.60 1.3 5229.0 100.0

7. 3.5 0.55 4.6 0.58 385.7 100.00 0.60 0.8 5990.0 100.0



100

PEAK DISPLAY (10µl of Sample)

PEAK DISPLAY (20µl of Sample) PEAK DISPLAY (30µl of Sample)

PEAK DISPLAY (40µl of Sample) PEAK DISPLAY (50µl of Sample)

Figure 3: Chromatogram of Abelmoschus esculentus. (L) flower extract (10μl to 50 μl of

sample)



101

Table 2: Shows peak table with Rf values, height and area of sample in methanol extract

of Abelmoschus esculentus. (L) flower

Peak Sample

(µl )

Start

Rf

Start

Height

Max

Rf

Max

Height

Height

%

End

Rf

End

Hei

ght

Area Area

%

1. 10 0.56 0.6 0.62 221.2 100.00 0.64 0.6 4091.4 100.0

2. 20 0.56 0.5 0.61 290.1 100.00 0.64 0.2 5689.3 100.0

3. 30 0.56 0.3 0.16 316.3 100.00 0.64 0.2 6683.7 100.0

4. 40 0.55 0.1 0.60 318.8 100.00 0.63 0.6 6839.2 100.0

5. 50 0.55 1.0 0.60 319.1 100.00 0.63 1.6 7084.8 100.0

Figure 4: 3D display of HPTLC chromatogram of flower extracts of Abelmoschus

esculentus. (L)

TABLE 3: COMPOUNDS IDENTIFIED BY LC-MS

Sr. No. R.T (min) Compound [M _ H] [M + H]

1 11.5-11.7 PROTOCATECHUIC ACID-4-O-

GLUCOSIDE 315.1 -

2 19.8-19.9 KAEMPFEROL-3-O-HEXOSIDE 447.1 -

3 19.9-20.3 I SORHAMNETIN-3-O-GLUCOSIDE 477.1 -

4 24.0-24.1 QUERCETIN DIMER 603.1 -

5 26.6-26.9 QUERCETIN 301.0 -



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FIGURE 5: Total Ion Chromatogram obtained from LCMS (Negative mode)

FIGURE 6: MSMS obtained from LCMS

PROTOCATECHUIC ACID-4-O- GLUCOSIDE



103

FIGURE 7: KAEMPFEROL-3-O-HEXOSIDE

FIGURE 8: ISORHAMNETIN-3-O-GLUCOSIDE



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FIGURE 9: QUERCETIN DIMER

FIGURE 10: QUERCETIN

The methanolic extract was subjected to LCMS analysis. Secondary metabolites present in

methanol extracts of Abelmoschus esculentus (L) flower such as terpenoids, saponins,

flavonoids, tannins, steroids and alkaloids were identified with the help of this technique. The

active principles with their molecular weight, retention time and structure are presented in

Table 3. The chromatogram and the double mass spectrum of the methanolic extract of the

test drug are shown in Figure 5 to 10.

The methanolic extract was subjected to LCMS analysis to understand the major molecules

present in the selected plant. In the methanol extracts of Abelmoschus esculentus (L) flower

molecules such as Protocatechuic Acid-4-o-Glucoside, Kaempferol-3-o-Hexoside,

Isorhamnetin-3-0- Glucoside, Quercetin Dimer and Quercetin derivative were identified.



105

DISCUSSION

In HPTLC profile, each and every metabolite has played specific role and function in

harmony with other metabolites within the organization framework of the cells in the defense

mechanism of the plants. Chromatographic fingerprinting of phytoconstituents can be used

for the assessment of quality consistency and stability of herbal extracts or products by

visible observation and comparison of the standardized fingerprint pattern [9]. WHO has

emphasized the need to ensure the quality of medicinal plant products by using modern

controlled techniques and applying suitable standards [10].

HPTLC profile was carried out as it could be used as a chemical standard to detect the

genuineness of the selected plant drug. The pharmaceutical and nutraceutical industries are

nowadays confronted with adulteration and cheating[11]. Determination of salient standards

for herbal drug is inevitable in this field to check adulteration and substitution. HPTLC

fingerprinting observed in the present study could serve as a chemical standard to check the

quality and genuineness of the selected plant drug.

As mint showed highest concentration of quercetin it can be used as good sources of

quercetin. Herbs are enormously important in both traditional and western medicine[12].

Quercetin is useful in some of the allergies such as hay fever, hives. It inhibits the production

and release of histamine and other allergic/inflammatory substances possibly by stabilizing

cell membranes of mast cells [13,14]. Quercetin is xanthine oxidase inhibitory by nature and

hence prevents the production of uric acid, thereby easing the gout symptoms [15,16].

Liquid Chromatography -Mass Spectrometry (LC-MS) is proved to be a very useful

technique for plant metabolite profiling and allows the identification of a large variety of

common plant metabolites in a single chromatogram. The research reveals the potential of

Abelmoschus esculentus (L) flower as a good source of bioactive compounds such as

Protocatechuic Acid-4-O- Glucoside, Kaempferol-3-O-Hexoside, Isorhamnetin-3-O-

Glucoside, Quercetin Dimer and Quercetin derivatives that justify the use of this plant for its

various ailments by traditional practitioners [17,18].

CONCLUSION

The HPTLC fingerprinting analysis showed that quercetin is present within the methanol

extract of Abelmoschus esculentus (L) flower. HPTLC fingerprinting observed in the present



106

study could serve as a chemical standard to check the quality and genuineness of the selected

plant drug. The methanolic extracts of Abelmoschus esculentus (L) flower confirmed the

presence of molecules such as Protocatechuic Acid-4-o-Glucoside, Kaempferol-3-o-

Hexoside, Isorhamnetin-3-0- Glucoside, Quercetin Dimer and Quercetin derivatives by

LCMS analysis.

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